PROJECT SUMMARY Breast cancer is the most common malignancy among women, with almost 1.7 million patients diagnosed annually. Although promising treatment options are currently available, metastatic disease remains a significant challenge. Indeed, the vast majority of breast cancer deaths are a direct consequence of metastasis and no cure is available once metastatic disease has developed. Thus, new treatment strategies are needed to eliminate mortality associated with metastatic breast cancer. This proposal is focused on the development of nanoparticle-mediated histotripsy (NMH) as a non-invasive and targeted ablation method for the treatment of multi-focal breast tumors. Histotripsy is a non-invasive, non-ionizing, and non-thermal ultrasound ablation method that destroys tissue through the precise control of acoustic cavitation generated by high- pressure (>25-30 MPa) pulses. Histotripsy does not have the limitations of thermal ablation and can produce consistent ablation, even in near vessels. Additional benefits of histotripsy include high precision and real-time imaging guidance. However, although histotripsy shows promise as an improved ablation method for the treatment of primary tumors, this treatment method requires high ultrasound pressures and remains limited to tumors that can be identified and imaged prior to treatment, which is often not feasible in metastatic breast cancer patients with multiple tumor nodules. To address this need, our team recently invented NMH as a targeted ablation approach for treating multi-focal tumors by combining perfluorohexane (PFH) nanoparticles with histotripsy. NMH takes advantage of the significantly reduced cavitation threshold of these particles, allowing for histotripsy to be selectively generated only in regions containing the particles. In this proposal, we develop NMH for the selective ablation of metastatic breast tumors. In Aim 1 we engineer and characterize PFH ?nanocones? with a smaller size (~50nm), higher stability, and functionalized targeting capabilities compared to our previously tested particles. Aim 2 will test the in vitro feasibility of these PFH ?nanocones? for targeting and selectively ablating breast cancer cells in a tissue engineered 3D breast cancer model. Aim 3 will investigate the in vivo feasibility of NMH for the targeted ablation of metastatic breast tumors in an orthotopic mouse model. Together, these studies are expected to demonstrate the feasibility of NMH for multi-focal tumor ablation, which is essential to establishing the potential of this new technology. If successful, this work will lay the foundation for NMH as a targeted multi-focal cancer therapy capable of significantly improving the standard of care for cancer patients by increasing targeting specificity and treating tumors too small to detect by imaging.